Contactless mixing using modulated air jets

ABSTRACT

Disclosed is a device for contactlessly mixing fluid present on the upper surface of the slide, where the device comprises a first nozzle array and a second nozzle array, the first nozzle array adapted to impart a bulk fluid flow to the fluid present on the upper surface of the slide, and the second nozzle array adapted to impart at least a first regional fluid flow to at least a portion of the fluid present on the upper surface of the slide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of PCT Application No.PCT/EP2018/081669 filed on Nov. 19, 2018, which application claims thebenefit of the filing date of U.S. Provisional Patent Application No.62/589,234 filed on Nov. 21, 2017, the disclosure of which is herebyincorporated by reference herein in its entirety.

BACKGROUND OF THE DISCLOSURE

Many tissues do not retain enough color after processing to make theircomponents visible under a bright-field microscope. Accordingly, it iscommon practice to add color and contrast to tissue components bystaining the tissue with various reagents. In the past, the steps ofstaining a tissue sample for histological or cytological analysis wereperformed manually, a process that is inherently inconsistent.Inconsistent staining makes it difficult for a pathologist or othermedical personnel to interpret slides and to make comparisons betweendifferent samples. Thus, a number of devices and methods have beendescribed that serve to automate the staining process and reducestaining inconsistency.

Devices for automated staining, especially for high volume staining withtraditional reagents such as hematoxylin and eosin (H&E), are primarilyof a “dip and dunk” type, where racks of slides are automaticallylowered into and removed from a series of reagent baths. For example,U.S. Pat. No. 4,911,098 to Tabata describes an automated stainingapparatus, where microscope slides holding tissue specimens are dippedsequentially into a large number of chemical solution containers. Theslides are mounted vertically in a slide holder basket and a clamp thatengages and disengages the basket is used to move the slides fromsolution to solution. The clamp can include a mechanism to tilt thebasket, which aids in removing excess solution before the basket issubmerged in the next solution. Additional automated staining devices ofthe “dip and dunk” type are described in U.S. Pat. No. 5,573,727 toKeefe, U.S. Pat. No. 6,080,363 to Takahasi et al., U.S. Pat. No.6,436,348 to Ljungmann et al. and U.S. Patent Application PublicationNo. 2001/0019703, naming Thiem et al. as inventors.

Another type of automatic staining apparatus delivers fresh reagentsdirectly to individual slides. For example, U.S. Pat. No. 6,387,326 toEdwards et al. describes an apparatus for staining slides where slidesare expelled one at a time from a slide storage device and individuallytreated at various staining stations as they move along a conveyor belttransport apparatus. Additional devices for automatically stainingindividual slides are described in U.S. Pat. No. 6,180,061 to Bogen etal., PCT Publication WO 03/045560, naming Tseung et al. as inventors,and U.S. Patent Application Publication No. U.S. 2004/0052685 namingRichards et al. as inventors.

Efficient mixing of fluids is an important step in many industrial,chemical, and pharmaceutical methods, as well as in biotechnologicalapplications. The mixing at small scales is often a difficult task. Insome embodiments, molecular diffusion becomes the main mixing mechanism,which makes the overall process slow. Integration of active mixers isoften difficult, increases the costs of any such device, and introducescross-contamination between samples.

BRIEF SUMMARY OF THE DISCLOSURE

Disclosed herein are systems and methods for the contactless dispersion,replenishment, and/or mixing (collectively referred to as “mixing”herein) of one or more fluids on a substrate (e.g. a microscope slide).Applicant has discovered that the use of a plurality of streams of gasdirected to a fluid (e.g. a puddle of fluid) present on the surface of asubstrate (e.g. a planar support surface) provides fluid movement anddirection to predetermined areas of the fluid, thus enabling mixing,distribution, and/or replenishment of the fluid or components within thefluid. Applicant has also surprisingly discovered that the introductionof such directed streams (such as separate or discrete streams) of gasachieves bulk mixing of fluid as opposed to mere agitation or localmixing. Indeed, Applicant has discovered that the contactless mixers ofthe present disclosure enable improved mixing as compared with merelystirring fluids on a substrate with an induced vortex (e.g. mixing withthe contactless mixers of the present disclosure allows quicker and morethorough mixing). Moreover, the contactless mixers of the presentdisclosure allow fluid movement in multiple locations across the slidesuch that bubbles, if formed, could be displaced and/or eliminated. Incontrast, mere vortex mixing may cause bubbles to move to the center ofa fluid.

Applicant has also discovered that mixing fluids according to themethods described herein (i) reduces the risk of staining artifacts;(ii) allows for a uniform reagent concentration across the cells and/ortissue during staining, e.g. to mitigate the formation of lightlystaining areas e.g. light spots) or the formation of darkly stainingareas (e.g. dark spots); (iii) enables increased mixing frequency; (iv)increases mixing efficacy; and/or (v) reduces or eliminates the presenceof bubbles in the fluids. It is also believed that the use of thedevices and methods disclosed herein may permit the use of lowerconcentrations of detection probes (e.g. antibodies) or other reagentsin any histological or cytological staining procedure. In addition, theuse of the devices and methods allows for low manufacturing andmaintenance costs, reduced process times, and/or mitigation ofcontamination. These and other benefits will be described furtherherein.

In one aspect of the present disclosure is a device which enablescontactless mixing of a fluid on a substrate, the device configured todirect a plurality of streams of gas to the fluid and/or to the surfaceof the substrate such that at least two discrete fluid motions areimparted to portions of the fluid at different times, the two discretefluid motions enabling cross-mixing. In some embodiments, the device isconfigured such that the gas streams mitigate loss of fluid from thesubstrate or splashing of the fluid from the substrate to an adjacentsubstrate, while adequately mixing the fluid present on the substratesurface.

In another aspect of the present disclosure is an automated slideprocessing apparatus comprising at least one contactless mixer formixing fluid present on the upper surface of the slide, where thecontactless mixer includes a first nozzle set and a second nozzle set,where the first nozzle set is configured to impart a first motion to afirst portion of fluid present on the upper surface of the slide, andthe second nozzle is set configured to impart a second motion to atleast a second portion of the fluid present on the upper surface of theslide. In some embodiments, the second motion induces cross-mixing ofthe fluid. In some embodiments, the first and second motions areopposite motions. In some embodiments, the second nozzle set imparts asecond motion and a third motion, wherein the second and third motionsmay be the same or different, but where at least one of the second orthird motions is opposite the first motion. In some embodiments, the gasstreams enables the fluid, or any portion thereof, to move in asubstantially circular path.

In some embodiments, the first set of nozzles are operated independentlyand at different times from the second set of nozzles, i.e. the firstset of nozzles are operated exclusively from the second set of nozzles.In this way, the first nozzle set may impart a first motion at leastduring the operation of the first nozzle set; and wherein the secondnozzle set may impart at least a second motion at least during theoperation of the second nozzle set. The skilled artisan will appreciatethat the first nozzle set may comprise a plurality of nozzles, and eachof the plurality of nozzles of the first nozzle set are operatedconcurrently to effectuate the first fluid motion. By way of example, afirst nozzle set may comprise first and second nozzles directing gasstreams towards one area of the fluid, and third and fourth nozzlesconcurrently directing gas streams toward another area of the fluid,where the first and second nozzles may direct gas in a first directionand whereby the third and fourth nozzles may direct gas in a second,opposite direction. Of course, the second nozzle may be similarlyconfigured for operation in a like manner, but to effectuate at leastthe second motion. In some embodiments, the automated slide processingapparatus further includes a second contactless mixer, such as onehaving one or more discrete nozzle sets, each discrete nozzle setcapable of independent operation.

In some embodiments, the first portion is a majority of the fluidpresent on the slide. In some embodiments, the first motion is appliedto the entirety of the fluid. In some embodiments, the first motion isone of a clockwise or counter-clockwise stirring. In yet otherembodiments, the first motion is imparted to at least 60% of the fluid.In even further embodiments, the first motion is imparted to at least70% of the fluid. In yet further embodiments, the first motion isimparted to at least 80% of the fluid.

In some embodiments, second motion is imparted to at least two discreteportions of the fluid present on the upper surface of the slide. In someembodiments, the second motion is imparted to at least three discreteportions of the fluid present on the upper surface of the slide. In someembodiments, the second motion is imparted to a center portion of thefluid (e.g. a center third of the entirety of the fluid). In someembodiments, the second nozzle set is further adapted to impart a thirdmotion to two end portions of fluid (e.g. the first and last thirds ofthe entirety of the fluid), where the two end portions of fluid are eachadjacent to the center portion, and wherein the third motion is oppositethe second motion. In some embodiments, the first motion is acounter-clockwise stirring, the second motion is a clockwise stirring,and the third motion is a counter-clockwise stirring.

In some embodiments, the second nozzle set is configured such that thesecond motion is imparted to at least two different portions of thefluid, e.g. non-adjacent portions of the fluid. In some embodiments, thesecond nozzle set is further adapted to impart a third motion to acenter portion of the fluid. In some embodiments, the first motion is acounter-clockwise motion, the second motion is a clockwise motion, andthe third motion is a counter-clockwise motion; and wherein the secondmotion is imparted to two end portions of fluid located on either sideof the center portion.

In some embodiments, the at least one contactless mixer is positionedabove the upper surface of the slide. In some embodiments, the at leastone contactless mixer is positioned between 0.3 inches (7.62 mm) toabout 1.5 inches (38.1 mm) above the slide. In some embodiments, the atleast one contactless mixer is positioned substantially parallel to theupper surface of the slide. In some embodiments, the at least onecontactless mixer body is offset at a predetermined angle relative tothe upper surface of the slide (e.g. offset at an angle between about 1degree to about 20 degrees).

In some embodiments, the first and second nozzle sets are configured todirect streams of gas to predetermined positions on the upper surface ofthe slide. In some embodiments, the first and second nozzle sets areconfigured to direct streams of gas to predetermined positions on theupper surface of the slide at predetermined angles of incidence relativeto the slide surface. In some embodiments, an angle of incidence (e.g.the angle from a line perpendicular to a surface) of a gas streamrelative to the upper surface of the slide is between about 15 degreesand about 90 degrees. In some embodiments, the gas streams may bedirected to positions between a fluid on the slide and an edge of theupper surface of the slide.

In some embodiments, the first nozzle set includes between 2 and 6nozzles. In some embodiments, the first nozzle set includes between 4and 6 nozzles. In some embodiments, the nozzles of the first nozzle setare grouped in two parallel rows along the longitudinal axis of thecontactless mixer. In some embodiments, each nozzle in the first nozzleset independently has a specific slant angle and offset angle, as thoseterms are defined herein. In some embodiments, the second nozzle setincludes between 2 and 4 nozzles. In some embodiments, the nozzles ofthe second nozzle set are grouped in two substantially parallel rowsalong the longitudinal axis of the contactless mixer. In someembodiments, each nozzle in the second nozzle set independently have aspecific slant angle and offset angle.

In some embodiments, the automated slide processing apparatus is astaining apparatus. In some embodiments, the automated slide processingapparatus further includes a control system, the control system adaptedto independently operate (e.g. pulse at predetermined intervals and/orat predetermined frequencies) the first and second nozzle sets to impartthe first and second motions, and any additional motions if thecontactless mixer is so configured. In some embodiments, the automatedslide processing apparatus further includes a third nozzle set to impartyet an additional direction of motion to the fluid present on the uppersurface of the slide.

In another aspect of the present disclosure is a method of operating acontactless mixer, the contactless mixer including a first nozzle setand a second nozzle set, the first nozzle set adapted to impart a firstmotion to the fluid present on the upper surface of the slide, and thesecond nozzle set adapted to impart a second motion to at least aportion of the fluid present on the upper surface of the slide, whereinthe method includes operating the first nozzle set for a first timeperiod, and then subsequently operating the second nozzle set for asecond time period, provided that the first and second nozzle arrays arenot operated concurrently. In some embodiments, the first and secondnozzle sets are both operated at least once. In some embodiments, thefirst and second nozzle sets are both operated at least twice. In someembodiments, the first and second nozzle sets are communicativelycoupled to a control system having one or more sensors, the sensorsadapted to determine a degree of mixing during or following operation ofthe first and/or second nozzle sets.

In another aspect of the present disclosure is an automated slideprocessing apparatus comprising: (i) at least one fluid dispenserconfigured to dispense a fluid onto an upper surface of aspecimen-bearing slide; and (ii) a contactless mixer for mixing fluidpresent on the upper surface of the slide, where the contactless mixerincludes a first nozzle array and a second nozzle array, the firstnozzle array adapted to impart a bulk fluid flow to the fluid present onthe upper surface of the slide, and the second nozzle array adapted toimpart at least a first regional fluid flow to at least a portion of thefluid present on the upper surface of the slide. In some embodiments,the first regional fluid flow induces a cross-mixing within the fluid.In some embodiments, the automated slide processing apparatus is astaining apparatus, and wherein the fluid present on the upper surfaceof the slides includes a reagent, non-limiting examples of which includea staining reagent, a counterstaining reagent, or a wash reagent. Otherreagents and or fluids which may be present on the surface of the slideare known to those of ordinary skill in the art.

In some embodiments, the bulk fluid flow is one of a clockwise orcounter-clockwise motion, e.g. a stirring or other rotational movement.In some embodiments, the first regional fluid flow is the other of aclockwise or counter-clockwise motion. In some embodiments, the firstregional fluid flow is imparted to a center portion of the fluid presenton the upper surface of the slide. In some embodiments, the firstregional fluid flow is imparted to a center portion of the fluid presenton the upper surface of the slide, the center portion representing abouta third of the fluid present on the upper surface of the slide. Theskilled artisan will appreciate that the first regional fluid flow mayimparted in any portion of the fluid on the slide, provided that thefirst regional fluid flow, along with any additional imparted regionalfluid flows, enables a cross-mixing.

In some embodiments, the second nozzle array is further adapted toimpart a second regional fluid flow and a third regional fluid flow, thesecond and third regional fluid flows occurring at different two endportions of the fluid on the slide, the two end portions each beingadjacent to the center portion, and wherein the second and thirdregional fluid flows are opposite the first regional fluid flow. In someembodiments, the second regional fluid flow and the third regional fluidflow are imparted from the second nozzle array substantiallysimultaneously.

In some embodiments, wherein the automated slide processing apparatus isa staining apparatus, and wherein the fluid present on the upper surfaceof the slide includes, but is not limited to, a staining reagent, acounterstaining reagent, or a wash reagent.

In another aspect of the present disclosure is a method of operating acontactless mixer, the contactless mixer including a first nozzle arrayand a second nozzle array, the first nozzle array adapted to impart abulk fluid flow to a fluid (or portion thereof) present on the uppersurface of the slide, and the second nozzle array adapted to impart atleast a first regional fluid flow to at least a portion of a fluidpresent on the upper surface of the slide, wherein the method includesoperating the first nozzle array for a first time period (e.g. to inducea bulk fluid flow for at least a portion of the time in which the firstnozzle array is operated), and then operating the second nozzle arrayfor a second time period (e.g. to induce at least one regional flow forat least a portion of the time in which the second nozzle array isoperated), provided that the first and second nozzle arrays are notoperated concurrently. In some embodiments, one of the first or secondtime periods is a predetermined time period. In some embodiments, one ofthe first or second time periods is determined in real-time using afeedback mechanism configured to interpret a level or degree of mixingachieved during operation of the first and/or second nozzle arrays. Insome embodiments, the feedback mechanism includes one or more sensorscommunicatively coupled to a control system. In some embodiments, thefirst and second nozzle arrays are operated sequentially at least twice.In some embodiments, operation of the first nozzle array is alwaysfollowed by operation of the second nozzle array, even if the operationof the second nozzle array is for a fraction of the time period in whichthe first nozzle array was operated. In other embodiments, the operationof the contactless mixer may include: (i) operating the first nozzlearray; (ii) subsequently operating the second nozzle array; and (iii)subsequently operating the first nozzle array. In some embodiments, thesecond nozzle array is configured such that at least second and thirdregional motions are induced, and wherein the second and third regionalfluid motions are induced during operation of the second nozzle array,and both the second and third regional fluid motions occur atsubstantially the same time.

In another aspect of the present disclosure is an automated slideprocessing apparatus comprising: (a) at least one fluid dispenserconfigured to dispense a fluid onto an upper surface of aspecimen-bearing slide; and (ii) a contactless mixer for distributingfluid present on the upper surface of the slide, where the contactlessmixer includes a first nozzle array in fluidic communication with afirst plenum, wherein the first nozzle array includes a first set ofprimary nozzles directing a gas stream in a first direction and a secondset of primary nozzles directing a gas stream in a second direction; anda second nozzle array in fluid communication with a second plenum. Insome embodiments, the second nozzle array includes a first set ofsecondary nozzles directing a gas stream in third direction and a secondset of secondary nozzles directing a gas stream in a fourth direction.In some embodiments, the second nozzle array further includes additionalsets of secondary nozzles, each additional set of nozzles adapted todirect a gas stream in another direction.

In some embodiments, the first and second directions are opposite eachother. In some embodiments, the gas streams emanating from the first andsecond sets of primary nozzles are directed substantially along aperiphery of the fluid present on the upper surface of the slide, and atany angle of incidence relative to the upper surface of the slide (orfluid positioned thereon). In some embodiments, the gas streamsemanating from the first and second sets of primary nozzles are directedto a portion of the slide adjacent to fluid, and at any angle ofincidence relative to the upper surface of the slide (or fluidpositioned thereon).

In some embodiments, the gas stream emanating from the first set ofprimary nozzles is directed substantially along a first longitudinalaxis of the slide; and wherein the gas stream emanating from the secondset of primary nozzles is directed substantially along a secondlongitudinal axis of the slide (either to the fluid, to an area of theslide not containing fluid, or to both). In some embodiments, the gasstreams from the first and second primary nozzles form an angle ofincidence with the surface of the slide ranging from between about 20degrees to about 80 degrees. In some embodiments, the gas streamsemanating from the first and second sets of primary nozzles areindependently offset by up to +/−15 degrees relative to a longitudinalaxis of the slide.

In some embodiments, the first nozzle array imparts a bulk fluid motionto the fluid present on the upper surface of the slide. In someembodiments, each nozzle in the first set of secondary nozzles directs agas stream to a different position on the upper surface of the slide;and wherein each nozzle in the second set of secondary nozzles directs agas stream to yet other different positions on the upper surface of theslide. In some embodiments, the second nozzle array establishes at leasttwo regional fluid flows. In some embodiments, the second nozzle arrayestablishes at least three regional fluid flows.

In another aspect of the present disclosure is a method of processing aspecimen-bearing slide comprising: (i) depositing a first fluid on thespecimen-bearing slide; and (ii) uniformly distributing the depositedfirst fluid on the specimen-bearing slide, wherein the deposited firstfluid is distributed by introducing a first set of gas streams to thedeposited first fluid to effectuate a first fluid motion to a firstportion of the deposited first fluid for a predetermined first period oftime, and introducing a second set of gas streams to the deposited firstfluid to effectuate at least a second fluid motion to at least a secondportion of the deposited first fluid for a predetermined second periodof time. In some embodiments, the first and second motions impart across-mixing. In some embodiments, the first fluid motion is impartedwith a first nozzle array; and wherein the at least the second fluidmotion is imparted with a second nozzle array. In some embodiments, thefirst portion is a majority of the first fluid on the slide, and whereinthe first set of gas streams induce a bulk fluid motion. In someembodiments, the second portion is a center portion of the first fluidon the slide (e.g. a center portion of a puddle of fluid). In someembodiments, the at least the second motion is a regional fluid motionimparted to a center portion of the fluid on the slide. In someembodiments, the second motion induces cross-mixing. In someembodiments, the first fluid is a reagent. In some embodiments, thefirst fluid is a mixture having multiple components.

In another aspect of the present disclosure is a method of processing aspecimen-bearing slide comprising: (i) depositing a first reagent on thespecimen-bearing slide; and (ii) uniformly distributing the depositedfirst reagent on the specimen-bearing slide, wherein the deposited firstreagent is distributed by introducing a first set of pulsed gas jets tothe deposited first reagent to effectuate a first fluid motion to afirst portion of the deposited first reagent for a predetermined firstperiod of time, and introducing a second set of pulsed gas jets to thedeposited first reagent to effectuate at least a second fluid motion toat least a second portion of the deposited first reagent for apredetermined second period of time. In some embodiments, the firstportion of the deposited first reagent includes a majority of thedeposited first reagent. In some embodiments, the first fluid flow is abulk fluid flow. In some embodiments, the at least the second portion ofthe deposited first reagent is a center portion of the deposited firstreagent. In some embodiments, the first and second motions are in thesame direction. In some embodiments, the first and second motions are inopposite directions. In some embodiments, the first predetermined timeperiod ranges from about 2 seconds to about 10 seconds. In someembodiments, the second predetermined time period ranges from about 2seconds to about 10 seconds. In some embodiments, a first set of gasjets and a second set of gas jets are sequentially operated at least 2times each. In some embodiments, a first set of gas jets and a secondset of gas jets are sequentially operated at least 4 times each. In someembodiments, pulsing with either first or second sets of gas jets occursat a frequency ranging from about 4 Hz to about 20 Hz. In someembodiments, additional reagent is dispensed to the upper surface of theslide and mixing with the gas jets is performed again to mix the secondreagent.

In another aspect of the present disclosure, is a contactless mixerhaving a first nozzle array and a second nozzle array, the first nozzlearray adapted to impart one or more gas streams to the fluid and/or thesubstrate such that substantially the entirety of a fluid on a substrateis moved along a first substantially circular path (first “swirl”motion); and wherein the second nozzle array is adapted to impart one ormore gas streams to the fluid and/or the substrate such that threedifferent portions of the fluid on the substrate are moved along second,third, and forth substantially circular paths (first, second, and third“swirl” motions). In some embodiments, the second nozzle array isadapted to provide for a cross-mixing of the fluid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates a bottom view of a contactless mixer having firstand second nozzle arrays in accordance with some embodiments.

FIG. 1B illustrates a bottom view of an alternative contactless mixerhaving first and second nozzle arrays in accordance with someembodiments.

FIG. 2A illustrates a bottom view of a contactless mixer having firstand second nozzle arrays in accordance with some embodiments.

FIG. 2B illustrates a bottom view of an alternative contactless mixerhaving first and second nozzle arrays in accordance with someembodiments.

FIG. 3A illustrates a perspective view of a contactless mixer inaccordance with some embodiments.

FIG. 3B illustrates a perspective view of an alternative contactlessmixer in accordance with some embodiments.

FIG. 4A provides a side view of a contactless mixer having a nozzlearray configured to direct gas streams to the surface of a substrate inaccordance with some embodiments.

FIG. 4B provides a side view of an alternative contactless mixer havinga nozzle array configured to direct gas streams to the surface of asubstrate in accordance with some embodiments.

FIGS. 5A and 5B provide perspective views of a contactless mixer and gasstreams emanating from nozzles within the contactless mixer inaccordance with some embodiments.

FIGS. 6A and 6B illustrate regional fluid movements induced by a nozzlearray in accordance with some embodiments.

FIGS. 6C and 6D illustrate bulk fluid movements induced by a nozzlearray in accordance with some embodiments.

FIG. 7 illustrates a nozzle arrangement of a contactless mixer, whereintwo sets of nozzles are illustrated, each set of nozzles in fluidiccommunication with a separate plenum in accordance with someembodiments.

FIG. 8 illustrates alternative nozzle shapes for use in a contactlessmixer in accordance with some embodiments.

FIGS. 9A and 9B illustrate a contactless mixer having a plurality ofnozzles, each nozzle directing a gas stream to a pre-determined locationon the substrate (or fluid positioned on the substrate) in accordancewith some embodiments.

FIG. 10A illustrates a contactless mixture having a plurality of inletsand nozzles.

FIGS. 10B and 10C illustrate the mixing of a dye within a fluid, the dyebeing substantially uniformly distributed within the fluid afteroperation of the contactless mixer.

FIG. 11A illustrates a contactless mixture having a plurality of inletsand nozzles.

FIG. 11B illustrates the mixing of a dye within a fluid, the dye beingsubstantially uniformly distributed within the fluid after operation ofthe contactless mixture.

DETAILED DESCRIPTION Definitions

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. The term “includes” is defined inclusively, suchthat “includes A or B” means including A, B, or A and B.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

As used herein, the terms “comprising,” “including,” “having,” and thelike are used interchangeably and have the same meaning. Similarly,“comprises,” “includes,” “has,” and the like are used interchangeablyand have the same meaning. Specifically, each of the terms is definedconsistent with the common United States patent law definition of“comprising” and is therefore interpreted to be an open term meaning “atleast the following,” and is also interpreted not to exclude additionalfeatures, limitations, aspects, etc. Thus, for example, “a device havingcomponents a, b, and c” means that the device includes at leastcomponents a, b and c. Similarly, the phrase: “a method involving stepsa, b, and c” means that the method includes at least steps a, b, and c.Moreover, while the steps and processes may be outlined herein in aparticular order, the skilled artisan will recognize that the orderingsteps and processes may vary.

As used herein, the term “biological sample” or “tissue sample” refersto any sample including a biomolecule (such as a protein, a peptide, anucleic acid, a lipid, a carbohydrate, or a combination thereof) that isobtained from any organism including viruses. Other examples oforganisms include mammals (such as humans; veterinary animals like cats,dogs, horses, cattle, and swine; and laboratory animals like mice, ratsand primates), insects, annelids, arachnids, marsupials, reptiles,amphibians, bacteria, and fungi. Biological samples include tissuesamples (such as tissue sections and needle biopsies of tissue), cellsamples (such as cytological smears such as Pap smears or blood smearsor samples of cells obtained by microdissection), or cell fractions,fragments or organelles (such as obtained by lysing cells and separatingtheir components by centrifugation or otherwise). Other examples ofbiological samples include blood, serum, urine, semen, fecal matter,cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus,biopsied tissue (for example, obtained by a surgical biopsy or a needlebiopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs(such as buccal swabs), or any material containing biomolecules that isderived from a first biological sample. In certain embodiments, the term“biological sample” as used herein refers to a sample (such as ahomogenized or liquefied sample) prepared from a tumor or a portionthereof obtained from a subject.

As used herein, the phrase “bulk fluid flow” refers to a motion impartedto a majority of a fluid on a substrate. Bulk fluid flow may be along acircular path. Bulk fluid flow may be in a specific direction, e.g.clockwise or counter-clockwise.

As used herein, the term “fluid” refers to any liquid, including water,solvents, solutions (e.g. buffer solutions), etc. The term “fluids” alsorefers to any mixtures, colloids, suspensions, etc. The term “fluids”also encompasses reagents, stains, and other specimen processing agents(e.g. glues, fixatives, etc.) which may be applied to a microscope slideand/or specimen. The fluids may be aqueous or non-aqueous. Furtherexamples include solutions or suspensions of antibodies, solutions orsuspensions of nucleic acid probes, and solutions or suspensions of dyeor stain molecules (e.g., H&E staining solutions, Pap stainingsolutions, etc.). Still further examples of fluids include solventsand/or solutions for deparaffinizing paraffin-embedded biologicalspecimens, aqueous detergent solutions, and hydrocarbons (e.g., alkanes,isoalkanes and aromatic compounds such as xylene). Still furtherexamples of fluids include solvents (and mixtures thereof) used todehydrate or rehydrate biological specimens.

As used herein, the terms “nozzle array”, “nozzle set” and “set ofnozzles” each refer to a series of nozzles which together are adapted toimpart a fluid flow, such as a bulk fluid flow or a regional fluid flow.In some embodiments, a “nozzle array” may itself include multiple “setsof nozzles,” or at least two series of nozzles.

As used herein, the term “plurality” refers to two or more, for example,3 or more, 4 or more, 5 or more, etc.

As used herein, the terms “reagent” refers to any liquid or liquidcomposition used in a specimen processing operation that involves addingliquid or liquid composition to a slide. Examples of reagents andprocessing liquids include solutions, emulsions, suspensions, andsolvents (either pure or mixtures thereof). These and other examples canbe aqueous or non-aqueous. Further examples include solutions orsuspensions of specific-binding entities, antibodies, solutions orsuspensions of nucleic acid probes, and solutions or suspensions of dyeor stain molecules (e.g., H&E staining solutions, Pap stainingsolutions, etc.). Still further examples include solvents and/orsolutions for deparaffinizing paraffin-embedded biological specimens,aqueous detergent solutions, and hydrocarbons (e.g., alkanes, isoalkanesand aromatic compounds such as xylene).

As used herein, the phrase “regional fluid flow” refers to a flow offluid in a particular portion or area of a fluid on a substrate, butless than a majority of the fluid on the substrate. A regional fluidflow may be in a specific direction, e.g. clockwise orcounter-clockwise. The skilled artisan will appreciate that any fluidpresent on the substrate may be divided into several areas, regions, orportions, a regional fluid flow may be imparted to any of these areas,regions, or portions.

As used herein, the term “slide” refers to any substrate (e.g.,substrates made, in whole or in part, glass, quartz, plastic, silicon,etc.) of any suitable dimensions on which a biological specimen isplaced for analysis, and more particularly to a “microscope slide” suchas a standard 3 inch by 1 inch microscope slide or a standard 75 mm by25 mm microscope slide. Examples of biological specimens that can beplaced on a slide include, without limitation, a cytological smear, athin tissue section (such as from a biopsy), and an array of biologicalspecimens, for example a tissue array, a cellular array, a DNA array, anRNA array, a protein array, or any combination thereof. Thus, in oneembodiment, tissue sections, DNA samples, RNA samples, and/or proteinsare placed on a slide at particular locations. In some embodiments, theterm slide may refer to SELDI and MALDI chips, and silicon wafers.

As used herein, the terms “stain,” “staining,” or the like as usedherein generally refers to any treatment of a biological specimen thatdetects and/or differentiates the presence, location, and/or amount(such as concentration) of a particular molecule (such as a lipid,protein or nucleic acid) or particular structure (such as a normal ormalignant cell, cytosol, nucleus, Golgi apparatus, or cytoskeleton) inthe biological specimen. For example, staining can provide contrastbetween a particular molecule or a particular cellular structure andsurrounding portions of a biological specimen, and the intensity of thestaining can provide a measure of the amount of a particular molecule inthe specimen. Staining can be used to aid in the viewing of molecules,cellular structures and organisms not only with bright-fieldmicroscopes, but also with other viewing tools, such as phase contrastmicroscopes, electron microscopes, and fluorescence microscopes. Somestaining performed by the system 2 can be used to visualize an outlineof a cell. Other staining performed by the system 2 may rely on certaincell components (such as molecules or structures) being stained withoutor with relatively little staining other cell components. Examples oftypes of staining methods performed by the system 2 include, withoutlimitation, histochemical methods, immunohistochemical methods, andother methods based on reactions between molecules (includingnon-covalent binding interactions), such as hybridization reactionsbetween nucleic acid molecules. Particular staining methods include, butare not limited to, primary staining methods (e.g., H&E staining, Papstaining, etc.), enzyme-linked immunohistochemical methods, and in situRNA and DNA hybridization methods, such as fluorescence in situhybridization (FISH).

As used herein, the term “substantially” means the qualitative conditionof exhibiting total or near-total extent or degree of a characteristicor property of interest. In some embodiments, “substantially” meanswithin about 20%. In some embodiments, “substantially” means withinabout 15%. In some embodiments, “substantially” means within about 10%.In some embodiments, “substantially” means within about 5%.

Devices and Systems

One aspect of the present disclosure is a contactless mixer deviceconfigured to introduce gas, such as pulses of a gas, to a fluid on thesurface of a substrate, e.g. a specimen-bearing microscope slide, toeffectuate mixing of one or more components within the fluid (e.g. apuddle of fluid on the upper surface of the microscope slide). In someembodiments, the substrate or microscope slide includes a biologicalsample comprising cells and/or tissue, and mixing by means of the pulsesof gas occurs without damaging the cells and/or tissue within the sampleand/or with minimal waste, i.e. the pulses of gas operate so as tominimize the amount of fluids directed off the surface of the substrate.

In some embodiments, the gas streams or pulses of gas are generated witha contactless mixer device, the contactless mixer including a pluralityof nozzles adapted to direct gas streams or pulses of gas to differentareas (e.g. predetermined areas, predetermined positions atpredetermined angles) on the surface of the substrate. In doing so, thedirected gas streams enable any fluid present on the slide to vibrate,move, and/or stir. As will be made described further herein, theplurality of nozzles are fluidically connected to a plenum, the plenumbeing in further communication with an inlet. The contactless mixer mayinclude one or more plenums, each plenum in fluidic communication with adifferent inlet, and each plenum further in communication with differentsets of nozzles.

With reference to FIGS. 1A-1B and 2A-2B, the contactless mixer 10 mayinclude one or more plenums 11A and 11B, each of the one or more plenumshaving plurality of nozzles 12A or 12B in fluidic communicationtherewith. In some embodiments, the one or more plenums 11A and 11B areeach independently in fluidic communication with an inlet 13A and 13B,respectively. In this way, a gas stream entering through the inlet 13Aor 13B may flow into the plenum 11A or 11B, respectively, where it isdistributed to each of the individual gas nozzles 12A or 12B andexpelled as a stream or jet of gas. While FIGS. 1A and 1B illustratethat the contactless mixer may comprise two sets of nozzles, each set ofnozzles in fluidic communication with a plenum 11A or 11B, which is inturn in communication with an inlet 13A or 13B, the contactless mixermay include only a single plenum, or may comprise more than two plenums.

Indeed, while the contactless mixer depicted in FIGS. 2A and 2B isillustrated a monolithic device, the skilled artisan will appreciatethat more than one contactless mixer may be positioned over a substrate,and each contactless mixer may include a plurality of nozzles in fluidiccommunication with one or more plenums and/or inlets. In this way, oneor more contactless mixers may be appropriately configured to direct gasstreams or pulses of gas to different areas on the surface of thesubstrate as detailed herein. For example, the systems herein mayinclude two or more contactless mixers, each contactless mixercomprising a single inlet in communication with a single plenum, whereinthe single plenum is in communication with a plurality of nozzles.

As illustrated in FIGS. 3A and 3B, in some embodiments the nozzles,plenums, and inlets are housed within a body 14, e.g. a monolithic bodyor a body comprised of multiple components, which is configurable forincorporation into a greater structure, e.g. a staining apparatus. thebody 14 may include one or more mounting or attachment points 15 suchthat the contactless mixer may be removably incorporated into a stainingapparatus or other instrument. In some embodiments, and as depicted inFIGS. 4A and 4B, the mounting or attachment points may be open at boththeir tops and bottoms, allowing for multiple points for connection toanother apparatus or instrument. The body 14 may be comprised of ametal, an alloy, a polymer, or a co-polymer. In some embodiments, thebody 14, the plenums, inlets, and nozzles may be fabricated by machininga solid block, molding, or otherwise securing multiple componentstogether to form the contactless mixer 10. In some embodiments, thecontactless mixer, and all components thereof, may be fabricated using3D printing.

In some embodiments, and with reference to at least FIGS. 1A and 1B, theplurality of nozzles 12A and 12B are arranged along a bottom surface 16of the contact less mixer 10. In some embodiments, the plurality ofnozzles is arranged in two or more rows. In some embodiments, thenozzles may be arranged parallel to each other, e.g. in rows parallelthe longitudinal axis 20 of the contactless mixer. In other embodiments,the nozzles may be arranged in a staggered configuration. In otherembodiments, the nozzles may be randomly configured in relation to oneanother. In embodiments where there are multiple sets of nozzles, eachset of nozzles may independently be configured. For example, FIG. 7illustrates a first set of nozzles (each individual nozzle labeled 12B)which are parallel to the longitudinal axis 20; and a second set ofnozzles (each individual nozzle labeled 12A) which are substantiallyparallel to the longitudinal axis 20.

The skilled artisan will appreciate that there exist many variableswhich may determine the flow rate of gas through any individual gasnozzle, including the size and shape of the nozzles, the number ofnozzles in fluidic communication with any individual plenum, not tomention the gas pressure supplied to the plenum through the inlet.

In some embodiments, each inlet is configured to receive a pressurizedgas from an external gas source (e.g. a pump, an air compressor, ablower, a fan, or other means sufficient to pressurize a gas from a gassource, if not already sufficiently pressurized). In some embodiments,the flow rate of a gas passing through each inlet independently rangesfrom about 1 L/min to about 5 L/min. In other embodiments, the flow rateof a gas passing through each inlet independently ranges from about 2L/min to about 25 L/min. Without wishing to be bound by any particulartheory, it is believed that the flow rate through each nozzle incommunication with the inlet is about the same.

The nozzles may have any size or shape. For example, the nozzles may becones (or substantially cone-shaped), helically rifled ports, smallarrays of angled orifices with small internal cone openings, or arrayswith uniform, round internal profiles and external bosses. Non-limitingexamples of such additional nozzle shapes are illustrated in FIG. 8. Insome embodiments, and regardless of the size or shape of the nozzles,the nozzles may be grouped together and, in some embodiments, thesimultaneous operation of a group of nozzles may enable individual gasstreams emanating from each individual nozzle to merge.

In some embodiments, the nozzles have an opening having a diameterranging from about 0.025 inches (0.635 mm) to about 0.035 inches (0.889mm). In other embodiments, the nozzles have an opening having a diameterranging from about 0.010 (0.254 mm) inches to about 0.030 inches (0.762mm). In yet other embodiments, the nozzles have an opening having adiameter of about 0.050 inches (1.27 mm). The skilled artisan willappreciate that the nozzles may each have the same or different diameteropenings.

In some embodiments, the plurality of nozzles are evenly spaced from oneanother. For example, considering a nozzle having a circular opening,the nozzles 12 may be spaced from about 0.150 inches (3.81 mm) to about0.300 inches (7.62 mm) apart from each other, as measured from thecenter of one circular opening to the center of another circularopening. In another example, and again assuming circular openings, thenozzles may be spaced from about 0.300 inches (7.62 mm) to about 0.500inches (12.7 mm) apart from each other. In yet another example, andagain assuming circular openings, the nozzles may be spaced from about1.00 inches (25.4 mm) apart from each other. Of course, the skilledartisan will appreciate that the nozzles may have any geometric shape,e.g. circular, ovoid, rectangular, square, etc.

The nozzles may be provided at various angles such that gas flow fromeach nozzle may be directed as desired (e.g. directed at a specificangle in any of the coordinate directions toward the surface of asubstrate) (see, for example, FIGS. 4A and 4B). The skilled artisan willappreciate that if any nozzle were to be considered as a single point,the nozzle may be formed within the body of the contactless mixer at anyangle and in any direction (x, y, or z) such that a stream or jet of gasmay be emanated from the nozzle (12A or 12B) and target the substratesurface (70) at a predetermined position at a predetermined angle ofincidence (see, for example, FIGS. 9A and 9B). The skilled artisan willfurther appreciate that by combining nozzles having different angles inany of the x, y, or z dimensions into an array, the various angles alongthe various coordinates will enable movement of fluid on a substrate ina direction dictated by the sets of nozzles.

FIGS. 5A and 5B illustrate two nozzle angles denoted as a “slant angle”and an “offset angle.” The skilled artisan will appreciate that bytuning the slant and offset angles of each nozzle independently, gasjets may be directed from the nozzle to different areas or positions(and at a particular entry angle) on a substrate positioned below thecontactless mixer. In general, a slant angle refers to the anglerelative to the substrate surface plane (i.e. a slant angle of 90degrees is straight down (normal to the surface); and a slant of 0degrees is parallel to the substrate surface). In some embodiments, theslant angle permits the gas streams emanating from the nozzles to imparta motion to a fluid on a substrate. In some embodiments, the slant anglemay be defined by considering the angle of incidence formed between agas stream emanating from a nozzle and the surface of a substratepositioned below the contactless mixer. In some embodiments, it isbelieved that the jets also interact with each other fluidically, makingthe actual incidence point on the substrate not “unpredictable,” butgoverned by a combination of compressible fluid dynamics and the nozzledirection. For example, in FIG. 5A, gas stream 30 has a 55-degree angleof incidence with the substrate. On the other hand, and as depicted inFIG. 5B, gas stream 31 has a 60-degree angle of incidence with thesubstrate.

In some embodiments, the slant angle ranges from between about 5 degreesto about 90 degrees. In other embodiments, the slant angle ranges frombetween about 10 degrees to about 70 degrees. In other embodiments, theslant angle ranges from between about 15 degrees to about 65 degrees. Inyet other embodiments, the slant angle ranges from between about 20degrees to about 60 degrees. In further embodiments, the slant angleranges from between about 25 degrees to about 55 degrees. In evenfurther embodiments, the slant angle ranges from between about 30degrees to about 55 degrees.

In addition to the slant angle, the nozzles may be defined by an offsetangle, which refers to an angle from which the gas nozzle deviates froman axis parallel to the longitudinal axis 20 or an axis parallel to thehorizontal axis 21 (sees FIGS. 5A and 5B). In some embodiments, theoffset angle permits the gas streams emanating from the nozzles toimpart directionality to a fluid on a substrate. In some embodiments,the offset angle ranges from between about 0 degrees to about 25degrees. In other embodiments, the offset angle ranges from betweenabout 5 degrees to about 15 degrees. In yet other embodiments, theoffset angle ranges from between about 10 degrees to about 15 degrees.

By way of another example, FIG. 5B depicts a gas stream 31 having aslant angle of 60 degrees relative to a plane parallel to the horizontalaxis 21, the gas stream also possessing an offset angle of 10 degreesrelative to an axis parallel the horizontal axis 21. Likewise, FIG. 5Adepicts a gas stream 30 having a slant angle of 55 degrees relative to aplane perpendicular to the longitudinal axis 20, the gas stream alsobeing offset by 5 degrees relative to an axis parallel the longitudinalaxis 20.

In the context of a microscope slide positioned beneath the contactlessmixer 10, in some embodiments a set of nozzles (e.g. between 4 and 12nozzles) are arranged such that they enable a bulk fluid flow to a fluidpositioned on the surface of the slide. In some embodiments, the jets ofgas emanating from some of the nozzles in the set of nozzles aredirected toward longitudinal edges of the slide or towards fluid nearthe longitudinal edges of the slide.

By way example, and with reference to FIGS. 6C and 6D, a first nozzlearray may be adapted to impart a bulk fluid motion 50 to the fluidpresent on the surface of a substrate. Here, nozzles in the first nozzlearray are arranged such that gas streams are directed substantiallytoward the edges 60 of a microscope slide, although not entirelyparallel to the edges 60. While the resulting bulk fluid motion depictedin FIGS. 6C and 6D is in a counter-clockwise direction, the skilledartisan will appreciate that the nozzle array may be configured suchthat the bulk fluid motion is in a clockwise direction. FIGS. 6A and 6Balso illustrates three discrete regional fluid flows 51, 52, and 53 or54, 55, and 56 where each of the discrete regional fluid flows areimparted with a second nozzle array configured to direct gas streams topredetermined positions on the surface of the slide at predeterminedangles of incidence. In FIG. 6A, regional fluid flows 51 and 53 aredepicted in a counter-clockwise direction, while regional fluid flow 52is in a clockwise direction. In FIG. 6B, Regional fluid flows 55 and 56are depicted in a clockwise direction, while regional fluid flow 54 isin a counter-clockwise direction. The skilled artisan will alsoappreciate that the nozzles within the second nozzle array may befurther adapted such that four or more regional fluid flows may beprovided, where each adjacent regional fluid flow is in a differentdirection.

Automated Slide Processing Systems

In another aspect of the present disclosure is an automated slideprocessing apparatus comprising at least one contactless mixerconfigured to introduce pulses of a gas to a fluid or a puddle on thesurface of the specimen-bearing microscope slide to effectuate mixing ofone or more components within the fluid or puddle. In some embodiments,the automated slide processing system includes at least one contactlessmixer having two discrete nozzle arrays, where each nozzle array isadapted to impart a flow to at least a portion of a fluid on the surfaceof a slide. In some embodiments, the automated slide processing systemincludes a contactless mixer having two discrete nozzle arrays, thefirst nozzle array adapted to impart bulk fluid flow in a firstdirection, and a second nozzle array adapted to impart at least oneregional fluid flow to at least one portion of the fluid, wherein the atleast one regional fluid flow is in a direction opposite the impartedbulk fluid flow. In some embodiments, the automated slide processingapparatus comprises at least two contactless mixers.

In some embodiments, specimen processing apparatus is an automatedapparatus, such as the BENCHMARK XT instrument, the SYMPHONY instrument,the BENCHMARK ULTRA instrument sold by Ventana Medical Systems, Inc.Ventana Medical Systems, Inc. is the assignee of a number of UnitedStates patents disclosing systems and methods for performing automatedanalyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809,6,352,861, 6,827,901 and 6,943,029, and U.S. Published PatentApplication Nos. 20030211630 and 20040052685, each of which isincorporated herein by reference in its entirety. Alternatively,specimens can be manually processed.

Examples of commercially available H&E stainers include the VENTANASYMPHONY (individual slide stainer) and VENTANA HE 600 (individual slidestainer) series H&E stainers from Roche; the Dako CoverStainer (batchstainer) from Agilent Technologies; the Leica ST4020 Small LinearStainer (batch stainer), Leica ST5020 Multistainer (batch stainer), andthe Leica ST5010 Autostainer XL series (batch stainer) H&E stainers fromLeica Biosystems Nussloch GmbH. The contactless mixer describes hereinmay be added to any of the aforementioned specimen processing systems.

The specimen processing apparatus can apply fixatives to the specimen.Fixatives can include cross-linking agents (such as aldehydes, e.g.,formaldehyde, paraformaldehyde, and glutaraldehyde, as well asnon-aldehyde cross-linking agents), oxidizing agents (e.g., metallicions and complexes, such as osmium tetroxide and chromic acid),protein-denaturing agents (e.g., acetic acid, methanol, and ethanol),fixatives of unknown mechanism (e.g., mercuric chloride, acetone, andpicric acid), combination reagents (e.g., Carnoy's fixative, methacarn,Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid),microwaves, and miscellaneous fixatives (e.g., excluded volume fixationand vapor fixation). The contactless mixer in fluidic communication withthe microscope slide may be used to uniformly distribute any of thesefixatives the slide or within another fluid, as detailed herein.

If the specimen is a sample embedded in paraffin, the sample can bedeparaffinized with the specimen processing apparatus using appropriatedeparaffinizing fluid(s). After the waste remover removes thedeparaffinizing fluid(s), any number of substances can be successivelyapplied to the specimen. The substances can be for pretreatment (e.g.,protein-crosslinking, expose nucleic acids, etc.), denaturation,hybridization, washing (e.g., stringency wash), detection (e.g., link avisual or marker molecule to a probe), amplifying (e.g., amplifyingproteins, genes, etc.), counterstaining, coverslipping, or the like.Again, any of these substances applied may be mixed or distributedthrough use of the contactless mixer described herein.

The specimen processing apparatus can apply a wide range of substancesto the specimen, which may then be uniformly distributed and/or mixedusing the contactless mixer in fluidic communication with the slideholder. The substances include, without limitation, stains, probes,reagents, rinses, and/or conditioners. The substances can be fluids(e.g., gases, liquids, or gas/liquid mixtures), or the like. The fluidscan be solvents (e.g., polar solvents, non-polar solvents, etc.),solutions (e.g., aqueous solutions or other types of solutions), or thelike. Reagents can include, without limitation, stains, wetting agents,antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.),antigen recovering fluids (e.g., aqueous- or non-aqueous-based antigenretrieval solutions, antigen recovering buffers, etc.), or the like.Probes can be an isolated nucleic acid or an isolated syntheticoligonucleotide, attached to a detectable label. Labels can includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes.

Automated IHC/ISH slide stainers typically include at least: reservoirsof the various reagents used in the staining protocols, a reagentdispense unit in fluid communication with the reservoirs for dispensingreagent to onto a slide, a waste removal system for removing usedreagents and other waste from the slide, and a control system thatcoordinates the actions of the reagent dispense unit and waste removalsystem. In addition to performing staining steps, many automated slidestainers can also perform steps ancillary to staining (or are compatiblewith separate systems that perform such ancillary steps), including:slide baking (for adhering the sample to the slide), dewaxing (alsoreferred to as deparaffinization), antigen retrieval, counterstaining,dehydration and clearing, and coverslipping. Prichard, Overview ofAutomated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp.1578-1582 (2014), incorporated herein by reference in its entirety,describes several specific examples of automated IHC/ISH slide stainersand their various features, including the intelliPATH (Biocare Medical),WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48(Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), LeicaBOND, and Lab Vision Autostainer (Thermo Scientific) automated slidestainers. Additionally, Ventana Medical Systems, Inc. is the assignee ofa number of United States patents disclosing systems and methods forperforming automated analyses, including U.S. Pat. Nos. 5,650,327,5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S.Published Patent Application Nos. 20030211630 and 20040052685, each ofwhich is incorporated herein by reference in its entirety.

Commercially-available staining units typically operate on one of thefollowing principles: (1) open individual slide staining, in whichslides are positioned horizontally and reagents are dispensed as apuddle on the surface of the slide containing a tissue sample (such asimplemented on the DAKO AUTOSTAINER Link 48 (Agilent Technologies) andintelliPATH (Biocare Medical) stainers); (2) liquid overlay technology,in which reagents are either covered with or dispensed through an inertfluid layer deposited over the sample (such as implemented on VENTANABenchMark and DISCOVERY stainers); (3) capillary gap staining, in whichthe slide surface is placed in proximity to another surface (which maybe another slide or a coverplate) to create a narrow gap, through whichcapillary forces draw up and keep liquid reagents in contact with thesamples (such as the staining principles used by DAKO TECHMATE, LeicaBOND, and DAKO OMNIS stainers). Some iterations of capillary gapstaining do not mix the fluids in the gap (such as on the DAKO TECHMATEand the Leica BOND). In variations of capillary gap staining termeddynamic gap staining, capillary forces are used to apply sample to theslide, and then the parallel surfaces are translated relative to oneanother to agitate the reagents during incubation to effect reagentmixing (such as the staining principles implemented on DAKO OMNIS slidestainers (Agilent)). In translating gap staining, a translatable head ispositioned over the slide. A lower surface of the head is spaced apartfrom the slide by a first gap sufficiently small to allow a meniscus ofliquid to form from liquid on the slide during translation of the slide.A mixing extension having a lateral dimension less than the width of aslide extends from the lower surface of the translatable head to definea second gap smaller than the first gap between the mixing extension andthe slide. During translation of the head, the lateral dimension of themixing extension is sufficient to generate lateral movement in theliquid on the slide in a direction generally extending from the secondgap to the first gap. See WO 2011-139978 A1. It has recently beenproposed to use inkjet technology to deposit reagents on slides. See WO2016-170008 A1. This list of staining technologies is not intended to becomprehensive, and any of the contactless mixers described herein may beused in conjunction with such systems to effectuate distribution andmixing of any fluids present on a specimen bearing microscope slide,including staining reagents.

In some embodiments is an apparatus for automatically treatingbiological specimens, comprising: at least one slide tray (such asdescribed herein) holding a plurality of slides in substantiallyhorizontal positions, wherein the biological specimens are located onthe slides; one or more workstations that receive the slide tray andperform one or more slide processing operations on the plurality ofslides held in the slide tray; a transporter that moves the slide trayinto and out of the one or more workstations; a fluidics module in fluidcommunication with the one or more workstations that supplies a reagentto the one or more workstations; a pneumatics module in fluidcommunication with the one or more workstations and the fluidics module;wherein the pneumatics module supplies vacuum and/or pressurized gas tothe one or more workstations and the fluidics module; a contactlessmixer posited above a slide in a slide tray to effectuate mixing; and acontrol module in electrical communication with the transporter, the oneor more workstations, the fluidics module and the pneumatics module,wherein the control module coordinates function of components (includingthe contactless mixer and its various components) of the apparatusduring treatment of the biological specimens.

In some embodiments, the apparatus for automatically treating biologicalspecimens further includes a control system for independentlycontrolling each contactless mixer such that mixing may be tailored foreach slide. In some embodiments, the apparatus for automaticallytreating biological specimens further includes one or more sensors orother feedback mechanisms to enable monitoring of the mixing and/ordistribution of the fluids dispensed onto the surface of the slide. Insome embodiments, the control system includes a microprocessor and oneor more microcontrollers, wherein the one or more microcontrollersreceive instructions from the microprocessor and separately control oneor more of the one or more workstations, the fluidics module, thecontactless mixer, and/or the transporter. In some embodiments, the atleast one of the workstations includes a moveable nozzle assembly,wherein the nozzle assembly includes one or more nozzles through whichthe reagent is delivered to a slide. The nozzles may be dispensenozzles.

In some embodiments, the workstation can perform a slide processingoperation on one or more individual slides in a slide tray, for example,at least two or four slides in a slide tray, or it can simultaneouslyperform a slide processing operation (including mixing operations withthe contactless mixer) on all of the slides in a slide tray. In someembodiments, one or more workstations dispense a reagent to slides inthe slide tray without a substantial amount of the reagent that contactsa first slide contacting a second slide, thereby minimizingcross-contamination between slides. Such workstations can include one ormore directional nozzles that dispense the reagent onto the slides, forexample, the one or more directional nozzles can include a pair ofdirectional nozzles that dispense the reagent in opposite directionsacross a surface of a slide. In more particular embodiments, the one ormore directional nozzles can further include a directional nozzle thatdispenses the reagent towards a bottom surface of a slide. In otherembodiments, the one or more workstations can simultaneously dispense areagent (for example, the same reagent) to at least two slides held in aslide tray within a given workstation, or the one or more workstationscan simultaneously dispense a reagent (such as the same reagent) to allof the slides held in the slide tray within a given workstation.Following dispensing of the fluids and or reagents, a contactless mixermay be independently activated to distribute and/or mix the fluids onthe surface of the slide.

In some embodiments is an automated method for processing a plurality ofslides bearing biological tissue samples, comprising: performing a setof slide processing operations on the plurality of slides in one or moreworkstations while the slides in the plurality are held in spatiallyco-planar, substantially horizontal positions in a slide tray, whereineach of the plurality of slides are positioned in proximity with atleast one contactless mixture; wherein the set of slide processingoperations includes at least staining samples on the slides in thespatially co-planar, substantially horizontal positions by flowing oneor more stains from at least one reagent container, through a fluidicsmodule, and out at least one dispense nozzle positioned above the slidetray, and solvent-exchanging; transporting the slide tray holding theplurality of slides to an automated coverslipper workstation afterperforming the set of slide processing operations that include at leaststaining and solvent-exchange; coverslipping the plurality of slidesheld in the slide tray with separate respective coverslips using theautomated coverslipper workstation while the plurality of slides areheld in spatially co-planar, substantially horizontal positions in theslide tray such that the coverslips on the slides are spaced apart fromone another; and removing the slide tray holding the coverslipped slidesfrom the automated coverslipper workstation. In some embodiments,processing includes the steps of (i) baking the samples under a radiantheater; (ii) de-paraffinizing the samples; (iii) staining the samples bydelivering one or more stains through one or more fluidic components andout one or more nozzles positioned generally above the slide tray,wherein the one or more fluidic components fluidically connect at leastone reagent container holding the one or more stains to the one or morenozzles; (iv) solvent-exchanging the samples; and (v) coverslipping thesamples with separate coverslips, wherein the aforementioned steps areautomatically performed by an apparatus comprising two or moreworkstations between which the slide tray holding the slides is movedduring processing.

In some embodiments, after the specimens are processed, a user cantransport specimen-bearing slides to the imaging apparatus. In someembodiments, the imaging apparatus is a brightfield imager slidescanner. One brightfield imager is the iScanCoreo™ brightfield scannersold by Ventana Medical Systems, Inc. In automated embodiments, theimaging apparatus is a digital pathology device as disclosed inInternational Patent Application No.: PCT/US2010/002772 (PatentPublication No.: WO/2011/049608) entitled IMAGING SYSTEM AND TECHNIQUESor disclosed in U.S. Patent Application Publication No. 2014/0178169,filed on Feb. 3, 2014, entitled IMAGING SYSTEMS, CASSETTES, AND METHODSOF USING THE SAME. International Patent Application No.PCT/US2010/002772 and U.S. Patent Application Publication No.2014/0178169 are incorporated by reference in their entities. In otherembodiments, the imaging apparatus includes a digital camera coupled toa microscope.

Control System

In some embodiments, the contactless mixer may be controlled with acontroller. In some embodiments, the control system may be incommunication with actuators, valves, and/or solenoids for controllingthe air entering the inlets of the contactless mixer, or for enablingpulsation of gas streams from each of the nozzles in fluidiccommunication with the inlets. In some embodiments, the control systemfurther includes a computer (including at least one processor and anon-tangible memory comprising instructions and for recordinginformation) that controls the opening and closing of actuators, valves,and/or solenoids such that nozzle arrays or sets of nozzles forming partof the contactless mixer may be operated (e.g. pulsed at certain timeintervals are certain frequencies). The control system permits dynamicadjustment of the actuators, values, and/or solenoids such that flow andpressure may be controlled along each plenum independently.

In some embodiments, the control system may further comprise one or moresensors for monitoring the gas jets directed toward the microscopeslide. In other embodiments, the control system may comprise one or morefeedback mechanisms to monitor mixing of the one or more fluids presenton the surface of the slide.

In some embodiments, the control system may further comprise one or moresensors for monitoring the surface tension, volume of fluid, temperatureof fluid and other mechanical properties of the fluid on the microscopeslide. In other embodiments, the control system may comprise one or morefeedback mechanisms to monitor the integrity of the fluid on the slideand dynamically adjust the actuators, values, and/or solenoids such thatflow and pressure may be controlled along each plenum independently toprevent or reduce forcing one or more fluids off the slide.

Optical or video detection and analysis can be employed to optimizemixing. By way of example, optical or video detection may be used todetect changes in color as a pigmented reagent mixes into a clear fluid.Other optical measurements such as spectral excitation, absorption,light scattering, fluorescence, luminescence, emission, polarizationmicroscopy, Raman scattering, and spectral analysis also can be used tomonitor mixing of fluids in contact with the sample on the surface ofthe microscope slide. The data may be acquired and analyzed by thecomputer or control system that is controlling the mixing process. Forexample, if sufficient mixing is achieved based on the received data,the contactless mixer may be turned off. By way of another example, ifmixing is insufficient based on the received data, the controller mayincrease the time in which the contactless mixer is turned on, or thecontroller may change one or more of the parameters associated withcontactless mixing, e.g. gas jet pulse frequency, gas pressure, or theset of nozzles delivering the gas.

Methods

The present disclosure also provides methods of mixing fluids present ona substrate, e.g. a slide, using the contactless mixers describedherein. By “mixed with the contactless mixer” it is meant that thecontactless mixer is operated to effectuate a mixing, distribution, orreplenishment of fluids within a puddle on a surface of a specimenbearing slide. As noted herein, the contactless mixer is configured suchthat pulses of gas (e.g. jets of gas or streams of gas) emanated from aplurality of nozzles or a series of sets of nozzles of the contactlessmixer impart a fluid flow (e.g. a stirring) to the fluid(s), causingmovement of the fluid in at least one direction in at least one area ofthe slide. As used herein, a “pulse” of gas may mean that a gas streamis “turned on” for a specific period of time, e.g. 1 second, and then“turned off.” This can be considered a continuous stream. Likewise, a“pulse” may also mean “turning on” a gas stream for a set period of timeduring which the gas is modulated at a particular frequency, e.g. a 1 Hzfrequency applied to a gas stream for a period of 5 seconds (i.e. thepulse may be a series of “turning on” and “turning off” for a set periodof time).

Therefore, another aspect of the present disclosure provides methods ofdistributing and/or mixing a fluid on the surface of a microscope slideby introducing pulses of gas to the puddle on the surface of thespecimen bearing slide. In some embodiments, introduction of the pulsedgas jets to the fluid causes movements and/or vibrations in the fluid,thus providing bulk fluid flow and/or regional fluid flows, ultimatelyenabling a substantially uniform distribution of a fluid over abiological sample. For example, and in some embodiments, a fluid may bedispensed to a predetermined area on the surface of a microscope slideand, upon activation of the contactless mixer and the introduction ofpulsed gas jets, the fluid may be distributed beyond the initial area ofdispensing. In some embodiments, the distribution of fluids on thesurface of the microscope slide through the introduction of pulsed gasjets may be used to facilitate the replenishment of fluids (e.g. areagent) on a biological sample mounted on the surface of the slide. Forexample, a biological sample may absorb a reagent deposited on itssurface (or may unevenly absorb a reagent) and eventually an amount ofreagent in contact with the biological sample may be substantiallydepleted (or depleted from a particular region or portion of thesample). Activation of the contactless mixer (or even individual nozzlearrays within the contactless mixer) may facilitate the distribution ofanother aliquot of the same reagent to the biological sample, thusreplenishing the reagent in contact with the biological sample.Activation of a contactless mixer (or individual nozzle arrays) may alsofacilitate the redistribution of the reagent from other areas of theslide to the biological sample faster than diffusional means, thusreplenishing the fluid or reagent in contact with the biological sample.

In other embodiments, a first fluid may already be present on thesurface of the microscope slide (e.g. a fluid puddle) and, followingintroduction of a second fluid, e.g. a reagent introduced via adispenser, the second fluid may be substantially uniformly distributedwithin the first fluid following the introduction of pulsed gas jetsfrom the contactless mixer. In some embodiments, by “substantiallyuniformly distributed,” it is meant that reagent concentration betweentwo separate points on the slide differ by no greater than 10% inmagnitude. In other embodiments, by substantially uniformly distributed,it is meant that reagent concentration between two separate points onthe slide differ by no greater than 5% in magnitude. In yet otherembodiments, by substantially uniformly distributed, it is meant thatreagent concentration between two separate points on the slide differ byno greater than 2% in magnitude. Of course, the skilled artisan willappreciate that any number of fluids may be deposited on the surface ofa microscope slide and each of those fluids may be mixed, i.e.substantially uniformly distributed within one another, through theintroduction of the pulsed air jets.

In some embodiments, the methods disclosed herein are suitable fordistributing and/or mixing any volume fluid on the surface of the slide.In some embodiments, the volume of fluid able to be distributed and/ormixed according to the method disclosed herein ranges from about 50 μLto about 2000 μL. In some embodiments, the volume of fluid able to bedistributed and/or mixed according to the method disclosed herein rangesfrom about 50 μL to about 1000 μL. In other embodiments, the volume offluid able to be distributed and/or mixed according to the methoddisclosed herein ranges from about 50 μL to about 750 μL. In yet otherembodiments, the volume of fluid able to be distributed and/or mixedaccording to the method disclosed herein ranges from about 50 μL toabout 500 μL. In yet other embodiments, the volume of fluid able to bedistributed and/or mixed according to the method disclosed herein rangesfrom about 100 μL to about 500 μL. The skilled artisan will be able toselect an appropriately configured contactless mixing nozzle array,including all operating parameters (e.g. the employment of specificnozzles having a particular configuration, air jet pulse frequency,time, pressure, and/or flow rate) such that the entire volume present onthe slide is substantially uniformly distributed and/or mixed asdesired.

Generally, the methods comprise (i) introducing a fluid to the surfaceof a slide; and (ii) introducing pulses of gas jets to the slide, thepulses of gas jets introducing fluid movements and/or vibrations to theslide. In some embodiments, the methods comprise additional stepsincluding, but not limited to, (a) detection steps for feedback controlof the contactless mixer, (b) fluid removal steps; and/or (c) additionalfluid dispensing steps.

In some embodiments, a method of processing specimen-bearing slidesincludes: (i) contacting a sample on the specimen-bearing slide with afirst reagent; and (ii) uniformly distributing the first reagent on thespecimen-bearing by introducing pulsed air jets to the specimen-bearingslide. As noted herein, the pulsed air jets may be delivered to thepuddle through an appropriately configured contactless mixer, such asone described herein that facilities at least bulk mixing of the fluidsor reagents within the puddle and/or regional mixing of fluids orreagents. In some embodiments, pulsed jets of air are emanated from afirst nozzle array and a second nozzle array of the contactless mixer.In some embodiments, the pulsed jets of air from the first and secondnozzle arrays are staggered in timing, thus allowed for alternatingperiods of bulk fluid flow and regional fluid flows. In someembodiments, the uniform distribution permits advancement of fluid toareas of the slide devoid of the fluid.

In some embodiments, the reagent is introduced into a first fluid puddleon the surface of the slide (e.g. a puddle comprising a buffer) or inproximity to a first puddle on the slide. In some embodiments, thereagent is detection probe. In some embodiments, the detection probesare binding moieties specific for a particular target within thebiological sample. In some embodiments, the detection probes utilizedare primary antibodies, namely primary antibodies which enable detectionof protein targets (or epitopes of protein targets) within the sample.In some embodiments, the primary antibody is conjugated to a detectablelabel, such as a fluorophore, a hapten, or an enzyme. In otherembodiments, the detection probes are nucleic acid probes which enabledetection of nucleic acid sequence targets within the sample. In otherembodiments, the specific binding moieties are nucleic acid probes,where the nucleic acid probes are conjugated to a detectable label, suchas a fluorophore, a hapten, or an enzyme.

In some embodiments, pulsing with any of the nozzles occurs at afrequency ranging from between about 0.5 Hz to about 15 Hz. In someembodiments, the acoustic source operates at a frequency ranging frombetween about 0.5 Hz to about 10 Hz. In some embodiments, pulsing withany of the nozzles occurs at a frequency ranging from between about 1 Hzto about 10 Hz. In some embodiments, pulsing with any of the nozzlesoccurs at a frequency ranging from between about 1 Hz to about 20 Hz.

In some embodiments, a first set of nozzles may operate at a firstfrequency or a first range of frequencies; while a second set of nozzlesmay operate at a second frequency or a second range of frequencies.Likewise, any set of nozzles of the contactless mixer may operate for afirst period of time at a first frequency, and then operate for a secondperiod of time at a second frequency. In other embodiments, a particularset of nozzles of the contactless mixer may operate initially at a firstfrequency, and the frequency may be increased or decreased over time(e.g. ramped up or ramped down over time at a predetermined interval).For example, a first frequency may be 10 Hz and a second frequency maybe 20 Hz, and the frequency may be ramped from 10 Hz in 1 Hz incrementsevery 0.5 seconds until the 20 Hz frequency is attained. In otherembodiments, the pneumatic source employs frequency modulation, wherebythe frequency is deviated by a value which is +/−20% of a predeterminedfrequency value.

In some embodiments, following introduction of the reagent, gas jetpulses are delivered to the puddle from at least one nozzle array of thecontactless mixer to effectuate at least bulk fluid mixing within thepuddle. In some embodiments, pulsed gas jets are introduced for a totaltime period ranging from between about 0.5 seconds to about 30 seconds.In some embodiments, pulsed gas jets are introduced for a time periodranging from between about 0.5 seconds to about 20 seconds. In otherembodiments, pulsed gas jets are introduced for a total time periodranging from between about 1 second to about 15 seconds. In otherembodiments, pulsed gas jets are introduced for a total time periodranging from between about 5 second to about 15 seconds. In otherembodiments, pulsed gas jets are introduced for a total time periodranging of about 10 seconds.

In some embodiments, the pulsing of the sample with gas jets may be atconstant intervals. For example, the sample may be pulsed with gas jetsfor a certain predetermined amount of time (e.g. 0.5 second intervals)followed by a predetermined amount of time in which no gas jets areintroduced (e.g. 1 second intervals). In other embodiments, the pulsingof the sample with gas jets may be at non-constant intervals. In otherembodiments, the determination as to whether the pulse the sample withgas jets may be determined by using a detector which provides feedbackas to the extent of mixing, or a detector which is able to detectwhether a portion of a slide or sample requires replenishment.

In some embodiments, the sample may be pulsed with pulsed gas jets forset periods during which a reagent is in contact with the sample (e.g.during an incubation period). For example, if an antibody is introducedto a sample and a protocol calls for the antibody to remain in contactwith the sample for a 360 second time period (e.g. an incubationperiod), gas jets may be introduced into the sample for a predeterminedamount of time at set intervals during the incubation period. Forexample, a pulse of gas may be introduced for 5 second time intervals attimes 0, +30 seconds, +60 seconds, +90 seconds, +120 seconds, +150seconds, +180 seconds, +210 seconds, +240 seconds, +270 seconds, +300seconds, and +330 seconds following introduction of the antibody. Ofcourse, rather than pulse the sample with gas jets at predeterminedintervals or for predetermined amounts of time, a feedback controldevice (such as described herein) may be utilized to determine whetherthe introduction of pulses is necessary, including the length of time inwhich the contactless mixer may be operated.

In embodiments where the contactless mixer includes multiple nozzlearrays, gas jets may be pulsed from each nozzle array sequentially. Forexample, gas jets may be pulsed from a first nozzle array for a firsttime period, followed by pulsing with gas gets from a second nozzlearray for a second time period. The sequential operation of the firstand second nozzle arrays may occur one or more times, e.g. between 1 and20 times. By way of example, gas jets within the first nozzle array maybe pulsed for a period of 5 seconds to impart a bulk fluid flow to apuddle present on the upper surface of the slide. Subsequently, pulsingwith gas jets from the second array for 5 seconds may impart regionalfluid flows to portions of the fluid present on the slide. In thisexample, the process of sequential operation may repeat 3 or more times.The skilled artisan will appreciate that each nozzle array may beindependently operated and each array may be pulsed for any period oftime to effectuate sufficient mixing.

In other embodiments, the slide or sample may be pulsed with gas from acontactless mixer (from any of one or more sets of nozzles or nozzlearrays) each time fluid is dispensed to the slide or sample and for anypurpose (i.e. fluid replenishment, fluid dispersing, and/or fluidmixing). For example, if a protocol calls for adding a certain aliquotof fluid every two minutes, pulses of gas may be supplied to the slideand/or sample for at least a predetermined amount of time each time analiquot is added. Of course, additional pulses may be supplied betweendispense cycles as needed.

In some embodiments, the method further includes detecting whether thefluid and/or reagent are adequately distributed or mixed (e.g. by usingthe feedback mechanisms described herein). If the detection stepdetermines that the fluid and/or reagent are not adequately mixed,operating parameters of the contactless mixer may be adjusted, e.g.pulse frequency, time, pressure, flow rate, and/or nozzle selection.

In addition, if a specific system or instrument calls for moving a slidebetween different stations or processing areas of the system orinstrument (e.g. a sample staining area, a sample incubation area), thecontactless mixer may be utilized to deliver pulsed jets of gas into thesample before and/or after movement of the slide to ensure that fluid isadequately distributed and/or mixed before, during, and after any suchmovements. Following mixing of the first reagent into the first fluidpuddle, the mixed first reagent/fluid puddle may be removed from thesurface of the slide. Subsequently, first detection reagents may beintroduced and then distributed on the surface of the slide or mixedwith a second fluid puddle existing on the slide. In some embodiments,the first detection reagents are specific for a label of the detectionprobe. For example, if the label is an enzyme, a substrate (a detectablemoiety, e.g. a chromogenic moiety) for the enzyme may be introduced suchthat a colored precipitate may be detected. In yet other embodiments, ananti-label antibody (a secondary antibody) is introduced to elicitdetection, where the anti-label antibody is specific to the label of theconjugate. For example, if the label is a hapten, an anti-haptenantibody specific to the hapten label is introduced, where theanti-hapten antibody includes a detectable moiety. In some embodiments,the detectable moiety of the anti-hapten antibody is an enzyme, and asubstrate for the enzyme is further introduced to detect the conjugateand target. The detectable moiety may then be detected according toprocesses known to those of ordinary skill in the art. The introductionof detection probes and/or detection reagents may be repeated “n” numberof times to account for any desired number of targets within the sample.

The methods disclosed herein are also suitable for multiplex assays. Forexample, a first detection probe specific to a first target and a seconddetection probe specific to a second target may be introducedsimultaneously or sequentially. Once both the first and second detectionprobes are introduced to the sample, the contactless mixer may beutilized to introduce pulses of gas to the sample such that the firstand second detection probes are mixed and uniformly distributed. Withoutwishing to be bound by any particular theory, it is believed that aneven distribution of the first and second detection probes mayfacilitate a uniform detection probe concentration during stainingand/or a reduction in staining artifacts. The skilled artisan willappreciate that any number of detection probes may be simultaneously orsequentially introduced to the sample on the surface of the slide, andthe “n” number of detection probes may be mixed with the contactlessmixer of the present disclosure. Following introduction of the detectionprobes, one or more detection reagents may be introduced, again eithersimultaneously and/or sequentially, and again mixed with the contactlessmixer.

In some embodiments, a method of replenishing a fluid or reagentincludes: (i) contacting a sample on the specimen-bearing slide with afirst reagent; (ii) allow time for the reagent to react with or beabsorbed by the sample; and (iii) uniformly distributing the firstreagent on the specimen-bearing by introducing pulses of gas to thespecimen-bearing slide from a contactless mixing apparatus, therebyreplenishing reagent to those areas that have been at least partiallydepleted of reagent. In some embodiments, the method optionally includesthe step of introducing additional aliquots of the first reagent priorto uniformly distributing the first reagent through the introduction ofthe pulses of gas. In some embodiments, the reagent is introduced into afirst fluid puddle on the surface of the slide (e.g. a puddle comprisinga buffer). In some embodiments, the reagent is detection probe. In someembodiments, the detection probes are binding moieties specific for aparticular target within the biological sample. In some embodiments, thedetection probes utilized are primary antibodies, namely primaryantibodies which enable detection of protein targets (or epitopes ofprotein targets) within the sample. In some embodiments, the primaryantibody is conjugated to a detectable label, such as a fluorophore, ahapten, or an enzyme. In other embodiments, the detection probes arenucleic acid probes which enable detection of nucleic acid sequencetargets within the sample. In other embodiments, the specific bindingmoieties are nucleic acid probes, where the nucleic acid probes areconjugated to a detectable label, such as a fluorophore, a hapten, or anenzyme.

In some embodiments, a method of processing specimen-bearing slidesincludes: (i) dispensing a first fluid onto a first portion of amicroscope slide; and (ii) distributing the first fluid on thespecimen-bearing by introducing pulses of gas to the specimen-bearingslide with a contact less mixer apparatus. In some embodiments, thefirst fluid is distributed from the first portion of the microscopeslide to at least a second portion of the microscope slide. In someembodiments, the first portion of the slide is a portion which does notcomprise a sample; and wherein the second portion of the slide includesa biological sample. In some embodiments, the fluid includes a detectionprobe. In some embodiments, the detection probes are binding moietiesspecific for a particular target within the biological sample. In someembodiments, the method further includes the step of introducingadditional aliquots of fluid to the slide (at any region), and thendistributing the fluid through the introduction of pulsed jets of gas.

EXAMPLES Example 1—Contactless Mixer

A nozzle array (see FIG. 10A) was developed to enable mixing in twodeliberate stages. During a first stage, the entire slide would be mixedin a single swirl; and then during a second stage, the slide would bedivided into three swirls for “cross mixing” the results of the singleswirl. Only two jets were used for each stage to simplify the design andreduce the chance for interference among the jets. This arrayillustrated the idea for whole slide and cross mixing. FIGS. 10B and 10Cillustrate the result of a dye being mixed with a fluid (e.g. a reactionbuffer) after operation of the contactless mixer.

Example 2—Contactless Mixer

A nozzle array (see FIG. 11A) was developed to enable mixing in twodeliberate stages. During a first stage, the entire slide would be mixedin a single swirl; and then during a second stage, the slide would bedivided into three swirls for “cross mixing” the results of the singleswirl. This array also illustrated the idea for whole slide and crossmixing. FIG. 11B illustrates the result of a dye being mixed with afluid (e.g. a reaction buffer) after operation of the contactless mixer.

Example 3—Methods of Pulsing

The nozzle arrays of a contactless mixer (e.g. the contactless mixer ofFIG. 10A or 11A) were pulsed using a high speed (2 ms response time)valve and a function generator using a modulation technique called“pulse width modulation” (PWM), for a set frequency and during eachperiod, the amount of “on time” vs. “off time” is varied. That ratio isreferred to as duty cycle. So, 20% duty cycle at any frequency means itis “on” 20% of the time, off 80% of the time. The frequency onlydetermines how often it switches on vs. off. For example, at 1 Hz, 80%duty cycle—the valve would turn on for 800 ms, off for 200 ms and repeatindefinitely. At 50% duty cycle and 1 Hz it would be on 500 ms, off 500ms. Either way the period is independent of duty cycle at 1000 ms andthe proportion of “on” vs. “off” time is what represents the duty cycle.So, for example at 20% duty cycle and 10 Hz, the valve would turn on 20ms, off 80 ms and repeat (100 ms period).

At 20% duty cycle, increasing the frequency to 8 or 16 Hz “evens out”the small pulses so they produce smaller waves (less agitation) and theshort duty cycle of 20% means it simply is not emitting much air. As thePWM frequency increases, it approaches similarity to a basic (non-PWM)air stream with 20% less air supply. At 50% duty cycle and any frequencythe results are generally less impressive than at 20 or 80% duty cycle.50% seems to be low enough to significantly reduce overall air flow, butnot low enough to enable standing-wave agitation and not high enough toprovide good bulk fluid movement.

At 80% duty cycle and any frequency the mixing is usually better than 20or 50%. 80% duty cycle and 4 Hz activation frequency sometimes providesa combination of agitation and bulk fluid movement, but at 16 Hz themixing is usually the best on almost all nozzles.

Based on these tests, it is believed that it would seem that bulk fluidmovement allows sufficient mixing across the entire slide—far moreimportant than localized agitation—and a steady, non-PWM nozzle willprovide the most bulk fluid movement.

Additional Embodiments and/or Components

In one aspect of the present disclosure is an automated slide processingapparatus comprising: (a) at least one fluid dispenser configured todispense a fluid onto an upper surface of a specimen-bearing slide; and(b) at least one contactless mixer for mixing fluid present on the uppersurface of the slide, where the contactless mixer includes a firstnozzle set and a second nozzle set, the first nozzle set configured toimpart a first motion to the fluid present on the upper surface of theslide, and the second nozzle set configured to impart a second motion toat least a portion of the fluid present on the upper surface of theslide (e.g. including, but not limited to, a portion of the fluid inwhich a first motion was induced). In some embodiments, the secondmotion induces cross-mixing of the fluid. In some embodiments, the firstset of nozzles are operated independently and at different times fromthe second set of nozzles, i.e. the first set of nozzles are operatedexclusive from the second set of nozzles. In some embodiments, theautomated slide processing apparatus includes a second contactlessmixer. In some embodiments, the first motion clockwise orcounter-clockwise. In some embodiments, the second motion is the otherof clockwise or counter-clockwise.

In another aspect of the present disclosure is an automated slideprocessing apparatus comprising: (i) a contactless mixer for mixingfluid present on the upper surface of the slide, where the contactlessmixer includes a first nozzle array and a second nozzle array, the firstnozzle array adapted to impart a bulk fluid flow to the fluid present onthe upper surface of the slide, and the second nozzle array adapted toimpart at least a first regional fluid flow to at least a portion of thefluid present on the upper surface of the slide. In some embodiments,the first regional fluid flow induces a cross-mixing within the fluid.In some embodiments, the automated slide processing apparatus is astaining apparatus, and wherein the fluid present on the upper surfaceof the slides includes a reagent, non-limiting examples of which includea staining reagent, a counterstaining reagent, or a wash reagent.

As noted herein, the automated slide processing apparatus may be tied toan imaging system. In some embodiments, the imaging system or apparatusmay be a multispectral imaging (MSI) system or a fluorescent microscopysystem. The imaging system used here is an MSI. MSI, generally, equipsthe analysis of pathology specimens with computerized microscope-basedimaging systems by providing access to spectral distribution of an imageat a pixel level. While there exists a variety of multispectral imagingsystems, an operational aspect that is common to all of these systems isa capability to form a multispectral image. A multispectral image is onethat captures image data at specific wavelengths or at specific spectralbandwidths across the electromagnetic spectrum. These wavelengths may besingled out by optical filters or by the use of other instrumentscapable of selecting a pre-determined spectral component includingelectromagnetic radiation at wavelengths beyond the range of visiblelight range, such as, for example, infrared (IR).

An MSI system may include an optical imaging system, a portion of whichcontains a spectrally-selective system that is tunable to define apre-determined number N of discrete optical bands. The optical systemmay be adapted to image a tissue sample, illuminated in transmissionwith a broadband light source onto an optical detector. The opticalimaging system, which in one embodiment may include a magnifying systemsuch as, for example, a microscope, has a single optical axis generallyspatially aligned with a single optical output of the optical system.The system forms a sequence of images of the tissue as the spectrallyselective system is being adjusted or tuned (for example with a computerprocessor) such as to assure that images are acquired in differentdiscrete spectral bands. The apparatus may additionally contain adisplay in which appears at least one visually perceivable image of thetissue from the sequence of acquired images. The spectrally-selectivesystem may include an optically-dispersive element such as a diffractivegrating, a collection of optical filters such as thin-film interferencefilters or any other system adapted to select, in response to either auser input or a command of the pre-programmed processor, a particularpass-band from the spectrum of light transmitted from the light sourcethrough the sample towards the detector.

An alternative implementation, a spectrally selective system definesseveral optical outputs corresponding to N discrete spectral bands. Thistype of system intakes the transmitted light output from the opticalsystem and spatially redirects at least a portion of this light outputalong N spatially different optical paths in such a way as to image thesample in an identified spectral band onto a detector system along anoptical path corresponding to this identified spectral band.

Embodiments of the subject matter and the operations described in thisspecification can be implemented in digital electronic circuitry, or incomputer software, firmware, or hardware, including the structuresdisclosed in this specification and their structural equivalents, or incombinations of one or more of them. For example, the control system maycomprise computer hardware and/or software, including any of thecomponents noted herein. Embodiments of the subject matter described inthis specification can be implemented as one or more computer programs,i.e., one or more modules of computer program instructions, encoded oncomputer storage medium for execution by, or to control the operationof, data processing apparatus. Any of the modules described herein mayinclude logic that is executed by the processor(s). “Logic,” as usedherein, refers to any information having the form of instruction signalsand/or data that may be applied to affect the operation of a processor.Software is an example of logic.

A computer storage medium can be, or can be included in, acomputer-readable storage device, a computer-readable storage substrate,a random or serial access memory array or device, or a combination ofone or more of them. Moreover, while a computer storage medium is not apropagated signal, a computer storage medium can be a source ordestination of computer program instructions encoded in an artificiallygenerated propagated signal. The computer storage medium can also be, orcan be included in, one or more separate physical components or media(e.g., multiple CDs, disks, or other storage devices). The operationsdescribed in this specification can be implemented as operationsperformed by a data processing apparatus on data stored on one or morecomputer-readable storage devices or received from other sources.

The term “programmed processor” encompasses all kinds of apparatus,devices, and machines for processing data, including by way of example aprogrammable microprocessor, a computer, a system on a chip, or multipleones, or combinations, of the foregoing. The apparatus can includespecial purpose logic circuitry, e.g., an FPGA (field programmable gatearray) or an ASIC (application-specific integrated circuit). Theapparatus also can include, in addition to hardware, code that createsan execution environment for the computer program in question, e.g.,code that constitutes processor firmware, a protocol stack, a databasemanagement system, an operating system, a cross-platform runtimeenvironment, a virtual machine, or a combination of one or more of them.The apparatus and execution environment can realize various differentcomputing model infrastructures, such as web services, distributedcomputing and grid computing infrastructures.

A computer program (also known as a program, software, softwareapplication, script, or code) can be written in any form of programminglanguage, including compiled or interpreted languages, declarative orprocedural languages, and it can be deployed in any form, including as astand-alone program or as a module, component, subroutine, object, orother unit suitable for use in a computing environment. A computerprogram may, but need not, correspond to a file in a file system. Aprogram can be stored in a portion of a file that holds other programsor data (e.g., one or more scripts stored in a markup languagedocument), in a single file dedicated to the program in question, or inmultiple coordinated files (e.g., files that store one or more modules,subprograms, or portions of code). A computer program can be deployed tobe executed on one computer or on multiple computers that are located atone site or distributed across multiple sites and interconnected by acommunication network.

The processes and logic flows described in this specification can beperformed by one or more programmable processors executing one or morecomputer programs to perform actions by operating on input data andgenerating output. The processes and logic flows can also be performedby, and apparatus can also be implemented as, special purpose logiccircuitry, e.g., an FPGA (field programmable gate array) or an ASIC(application-specific integrated circuit).

Processors suitable for the execution of a computer program include, byway of example, both general and special purpose microprocessors, andany one or more processors of any kind of digital computer. Generally, aprocessor will receive instructions and data from a read-only memory ora random-access memory or both. The essential elements of a computer area processor for performing actions in accordance with instructions andone or more memory devices for storing instructions and data. Generally,a computer will also include, or be operatively coupled to receive datafrom or transfer data to, or both, one or more mass storage devices forstoring data, e.g., magnetic, magneto-optical disks, or optical disks.However, a computer need not have such devices. Moreover, a computer canbe embedded in another device, e.g., a mobile telephone, a personaldigital assistant (PDA), a mobile audio or video player, a game console,a Global Positioning System (GPS) receiver, or a portable storage device(e.g., a universal serial bus (USB) flash drive), to name just a few.Devices suitable for storing computer program instructions and datainclude all forms of non-volatile memory, media and memory devices,including by way of example semiconductor memory devices, e.g., EPROM,EEPROM, and flash memory devices; magnetic disks, e.g., internal harddisks or removable disks; magneto-optical disks; and CD-ROM and DVD-ROMdisks. The processor and the memory can be supplemented by, orincorporated in, special purpose logic circuitry.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

Although the present disclosure has been described with reference to anumber of illustrative embodiments, it should be understood thatnumerous other modifications and embodiments can be devised by thoseskilled in the art that will fall within the spirit and scope of theprinciples of this disclosure. More particularly, reasonable variationsand modifications are possible in the component parts and/orarrangements of the subject combination arrangement within the scope ofthe foregoing disclosure, the drawings, and the appended claims withoutdeparting from the spirit of the disclosure. In addition to variationsand modifications in the component parts and/or arrangements,alternative uses will also be apparent to those skilled in the art.

Additional Embodiments Additional Embodiment 1

An automated slide processing apparatus comprising: at least one fluiddispenser configured to dispense a fluid onto an upper surface of aspecimen-bearing slide; and a contactless mixer for mixing fluid presenton the upper surface of the slide, where the contactless mixer comprisesa first nozzle set and a second nozzle set, the first nozzle set adaptedto impart a first motion to the fluid present on the upper surface ofthe slide, and the second nozzle set adapted to impart at least a secondmotion to at least a portion of the fluid present on the upper surfaceof the slide, wherein the at least the second motion enablescross-mixing of the fluid.

Additional Embodiment 2

The automated slide processing apparatus of additional embodiment 1,wherein the first motion is one of a clockwise or counter-clockwisestirring.

Additional Embodiment 3

The automated slide processing apparatus of any of the precedingadditional embodiments, wherein the second motion is the other of theclockwise or counter-clockwise stirring.

Additional Embodiment 4

The automated slide processing apparatus of additional embodiment 3,wherein the second motion is imparted to a center portion of the fluid.

Additional Embodiment 5

The automated slide processing apparatus of additional embodiment 4,wherein the second nozzle set is further adapted to impart a thirdmotion to two end portions of the fluid, the two end portions of thefluid each adjacent to the center portion, and wherein the third motionis opposite the second motion.

Additional Embodiment 6

The automated slide processing apparatus of additional embodiment 5,wherein the first motion is a counter-clockwise stirring, the secondmotion is a clockwise stirring, and the third motion is acounter-clockwise stirring.

Additional Embodiment 7

The automated slide processing apparatus of additional embodiment 3,wherein the second motion is imparted to at least two portions of thefluid.

Additional Embodiment 8

The automated slide processing apparatus of additional embodiment 7,wherein the second nozzle set is further adapted to impart a thirdmotion to a center portion of the fluid.

Additional Embodiment 9

The automated slide processing apparatus of additional embodiment 8,wherein the first motion is a counter-clockwise motion, the secondmotion is a clockwise motion, and the third motion is acounter-clockwise motion; and wherein the second motion is imparted totwo end portions of fluid located on either side of the center portion.

Additional Embodiment 10

The automated slide processing apparatus of any of the precedingadditional embodiments, wherein the contactless mixer is positionedabove the upper surface of the slide.

Additional Embodiment 11

The automated slide processing apparatus of additional embodiment 2,wherein the contactless mixer is positioned substantially parallel tothe upper surface of the slide.

Additional Embodiment 12

The automated slide processing apparatus of any of the precedingadditional embodiments, wherein the first and second nozzle sets areconfigured to direct streams of gas to predetermined positions on theupper surface of the slide.

Additional Embodiment 13

The automated slide processing apparatus of any of the precedingadditional embodiments, wherein the first nozzle set comprises between 2and 6 nozzles.

Additional Embodiment 14

The automated slide processing apparatus of additional embodiment 1,wherein the second nozzle set comprises between 2 and 4 nozzles.

Additional Embodiment 15

The automated slide processing apparatus of additional embodiment 5,wherein the nozzles of the first nozzle set are grouped in two parallelrows along the longitudinal axis of the contactless mixer.

Additional Embodiment 16

The automated slide processing apparatus of additional embodiment 5,wherein the nozzles of the second nozzle set are grouped in twosubstantially parallel rows along the longitudinal axis of thecontactless mixer.

Additional Embodiment 17

The automated slide processing apparatus of any of the precedingadditional embodiments, wherein the first motion is imparted to amajority of the fluid on the upper surface of the slide.

Additional Embodiment 18

The automated slide processing apparatus of additional embodiment 1,wherein the second motion is imparted to at least two discrete portionsof the fluid present on the upper surface of the slide.

Additional Embodiment 19

The automated slide processing apparatus of additional embodiment 1,wherein the second motion is imparted to at least three discreteportions of the fluid present on the upper surface of the slide.

Additional Embodiment 20

The automated slide processing apparatus of additional embodiment 1,wherein the first motion is one of a clockwise or a counterclockwisemotion; and wherein the second motion is opposite the first motion.

Additional Embodiment 21

The automated slide processing apparatus of any of the precedingadditional embodiments, wherein the automated slide processing apparatusis a staining apparatus, and wherein the fluid present on the uppersurface of the slides comprises a reagent selected from the groupconsisting of a staining reagent, a counterstaining reagent, or a washreagent.

Additional Embodiment 22

The automated slide processing apparatus of any of the precedingadditional embodiments, further comprising a control system, the controlsystem adapted to independently operate the first and second nozzle setsto impart the first and second motions.

Additional Embodiment 23

The automated slide processing apparatus of additional embodiment 22,wherein the control system includes one or more sensors for monitoring asurface tension, a volume of the at least one fluid, a temperature ofthe at least one fluid, or mechanical properties of the at least onefluid on a microscope slide.

Additional Embodiment 24

The automated slide processing apparatus of any of additionalembodiments 22 and 23, wherein the control system includes one or moresensors for monitoring streams of gas emanating from the first andsecond sets of nozzles.

Additional Embodiment 25

The automated slide processing apparatus of any of additionalembodiments 22 to 24, wherein the control system includes one or morefeedback mechanisms to monitor mixing.

Additional Embodiment 26

The automate slide processing apparatus of any of additional embodiments22 to 25, wherein the control system is adapted to dynamically adjustactuators, values, and/or solenoids to control a gas flow.

Additional Embodiment 27

The automated slide processing apparatus of any of the precedingadditional embodiments, further comprising a third nozzle set to impartan additional motion to the fluid present on the upper surface of theslide.

Additional Embodiment 28

An automated slide processing apparatus comprising: at least one fluiddispenser configured to dispense a fluid onto an upper surface of aspecimen-bearing slide; and a contactless mixer for mixing the fluidpresent on the upper surface of the slide, where the contactless mixercomprises a first nozzle array and a second nozzle array, the firstnozzle array adapted to impart a bulk fluid flow to the fluid present onthe upper surface of the slide, and the second nozzle array adapted toimpart at least a first regional fluid flow to at least a portion of thefluid present on the upper surface of the slide.

Additional Embodiment 29

The automated slide processing apparatus of additional embodiment 28,wherein the bulk fluid flow is in a clockwise or counter-clockwisedirection.

Additional Embodiment 30

The automated slide processing apparatus of additional embodiment 28 or29, wherein the first regional fluid flow is the other of the clockwiseor counter-clockwise direction.

Additional Embodiment 31

The automated slide processing apparatus of additional embodiment 30,wherein the first regional fluid flow is imparted to a center portion ofthe fluid present on the upper surface of the slide.

Additional Embodiment 32

The automated slide processing apparatus of additional embodiment 31,wherein the second nozzle array is further adapted to impart a secondregional fluid flow and a third regional fluid flow, the second andthird regional fluid flows occurring at two different end portions ofthe fluid on the slide, the two end portions each being adjacent to thecenter portion, and wherein the second and third regional fluid flowsare opposite the first regional fluid flow.

Additional Embodiment 33

The automated slide processing apparatus of any of additionalembodiments 28 to 32, wherein the automated slide processing apparatusis a staining apparatus, and wherein the fluid present on the uppersurface of the slides comprises a reagent selected from the groupconsisting of a staining reagent, a counterstaining reagent, or a washreagent.

Additional Embodiment 34

An automated slide processing apparatus comprising: at least one fluiddispenser configured to dispense a fluid onto an upper surface of aspecimen-bearing slide; and a contactless mixer for distributing fluidpresent on the upper surface of the slide, where the contactless mixerincludes a first nozzle array in fluidic communication with a firstplenum, wherein the first nozzle array comprises a first set of primarynozzles directing a gas stream in a first direction and a second set ofprimary nozzles directing the gas stream in a second direction; and asecond nozzle array in fluid communication with a second plenum, whereinthe second nozzle array comprises a first set of secondary nozzlesdirecting the gas stream in a third direction and a second set ofsecondary nozzles directing the gas stream in a fourth direction.

Additional Embodiment 35

The automated slide processing apparatus of additional embodiment 34,wherein the first and second directions are opposite each other.

Additional Embodiment 36

The automated slide processing apparatus of additional embodiment 35,wherein the gas streams emanating from the first and second sets ofprimary nozzles are directed substantially along a periphery of thefluid present on the upper surface of the slide.

Additional Embodiment 37

The automated slide processing apparatus of additional embodiment 35,wherein the gas stream emanating from the first set of primary nozzlesis substantially along a first longitudinal axis of the slide; andwherein the gas stream emanating from the second set of primary nozzlesis substantially along a second longitudinal axis of the slide.

Additional Embodiment 38

The automated slide processing apparatus of additional embodiment 37,wherein the gas streams from the first and second primary nozzles forman angle of incidence with the surface of the slide ranging from betweenabout 5 degrees to about 90 degrees.

Additional Embodiment 39

The automated slide processing apparatus of additional embodiment 37,wherein the gas streams emanating from the first and second sets ofprimary nozzles are independently offset by up to +/−15 degrees relativeto the longitudinal axis of the slide.

Additional Embodiment 40

The automated slide processing apparatus of additional embodiment 36,wherein the first nozzle array imparts a bulk fluid motion to the fluidpresent on the upper surface of the slide.

Additional Embodiment 41

The automated slide processing apparatus of additional embodiment 34,wherein each nozzle in the first set of secondary nozzles directs a gasstream to a different position on the upper surface of the slide; andwherein each nozzle in the second set of secondary nozzles directs a gasstream to a different position on the upper surface of the slide.

Additional Embodiment 42

The automated slide processing apparatus of additional embodiment 41,wherein the second nozzle array establishes at least two regional fluidflows.

Additional Embodiment 43

A method of processing a specimen-bearing slide comprising: (i)depositing a first reagent on the specimen-bearing slide; and (ii)uniformly distributing the deposited first reagent on thespecimen-bearing slide, wherein the deposited first reagent isdistributed by introducing a first set of pulsed gas jets to thedeposited first reagent to effectuate a first fluid motion to a firstportion of the deposited first reagent for a predetermined first periodof time, and introducing a second set of pulsed gas jets to thedeposited first reagent to effectuate at least a second fluid motion toat least a second portion of the deposited first reagent for apredetermined second period of time.

Additional Embodiment 44

The method of additional embodiment 43, wherein the first portion of thedeposited first reagent comprises a majority of the deposited firstreagent.

Additional Embodiment 45

The method of additional embodiment of additional embodiment 43 or 44,wherein the first fluid flow is a bulk fluid motion.

Additional Embodiment 46

The method of any of additional embodiments 43 to 45, wherein the atleast the second portion of the deposited first reagent is a centerportion of the deposited first reagent.

Additional Embodiment 47

The method of any of additional embodiments 43 to 46, wherein the firstand second motions are in the same direction.

Additional Embodiment 48

The method of any of additional embodiments 43 to 46, wherein the firstand second motions are in opposite directions.

Additional Embodiment 49

The method of any of additional embodiments 43 to 46, wherein a totalperiod of time for pulsing the first ranges from about 4 seconds toabout 120 seconds.

Additional Embodiment 50

The method of any of additional embodiments 43 to 46, wherein the firstpredetermined time period ranges from about 2 seconds to about 10seconds.

Additional Embodiment 51

The method of any of additional embodiments 43 to 46, wherein the secondpredetermined time period ranges from about 2 seconds to about 10seconds.

Additional Embodiment 52

The method of any of additional embodiments 43 to 46, wherein the firstset of gas jets and the second set of gas jets are sequentially operatedat least 2 times each.

Additional Embodiment 53

The method of any of additional embodiments 43 to 46, wherein pulsingwith either the first or second sets of gas jets occurs at a frequencyranging from about 4 Hz to about 20 Hz.

The invention claimed is:
 1. An automated slide processing apparatuscomprising: at least one fluid dispenser configured to dispense a fluidonto an upper surface of a specimen-bearing slide; and a contactlessmixer for distributing the fluid present on the upper surface of theslide, where the contactless mixer includes a first nozzle array influidic communication with a first plenum, wherein the first nozzlearray comprises a first set of primary nozzles directing a first gasstream in a first direction and a second set of primary nozzlesdirecting a second gas stream in a second direction; and a second nozzlearray in fluid communication with a second plenum, wherein the secondnozzle array comprises a first set of secondary nozzles directing athird gas stream in a third direction and a second set of secondarynozzles directing a fourth gas stream in a fourth direction, wherein thefirst and second directions are opposite each other and wherein the gasstreams emanating from the first and second sets of primary nozzles aredirected substantially along a periphery of the fluid present on theupper surface of the slide.
 2. The automated slide processing apparatusof claim 1, wherein the first nozzle array comprises between 2 and 6nozzles and wherein the second nozzle array comprises between 2 and 4nozzles.
 3. The automated slide processing apparatus of claim 1, furthercomprising a control system, the control system adapted to independentlyoperate the first and second nozzle arrays.
 4. The automated slideprocessing apparatus of claim 1, wherein the gas streams emanating fromthe first and second sets of primary nozzles form an angle of incidencewith the surface of the slide ranging from between about 20 degrees toabout 60 degrees.
 5. The automated slide processing apparatus of claim4, wherein the angle of incidence ranges from between about 25 degreesto about 55 degrees.
 6. The automated slide processing apparatus ofclaim 1, wherein the third and fourth directions are opposite eachother.
 7. The automated slide processing apparatus of claim 1, whereinthe gas streams emanating from the first and second sets of primarynozzles are independently offset by up to +/−15 degrees relative to thelongitudinal axis of the slide.
 8. The automated slide processingapparatus of claim 1, wherein the first nozzle array imparts a bulkfluid motion to the fluid present on the upper surface of the slide. 9.The automated slide processing apparatus of claim 1, wherein the firstand second gas streams are directed to different positions on the uppersurface of the slide; and wherein each nozzle in the second set ofsecondary nozzles directs the fourth gas stream to a different positionon the upper surface of the slide.
 10. The automated slide processingapparatus of claim 1, wherein the second nozzle array comprise a thirdset of secondary nozzles directing a fifth gas stream in a fifthdirection.
 11. The automates slide processing apparatus of claim 10,wherein the third and fifth directions are the same, and wherein thefourth direction is different from the third and fifth directions. 12.The automated slide processing apparatus of claim 1, wherein thecontactless mixer is positioned above and substantially parallel to theupper surface of the slide.
 13. The automated slide processing apparatusof claim 1, wherein the nozzles of the first nozzle array are grouped intwo parallel rows along the longitudinal axis of the contactless mixer.14. The automated slide processing apparatus of claim 1, wherein thenozzles of the second nozzle array are grouped in two substantiallyparallel rows along the longitudinal axis of the contactless mixer.